Novel defects in collagen XII and VI expand the mixed myopathy/Ehlers-Danlos syndrome spectrum and lead to variant-specific alterations in the extracellular matrix.

PURPOSE: To date, heterozygous or homozygous COL12A1 variants have been reported in 13 patients presenting with a clinical phenotype overlapping with collagen VI-related myopathies and Ehlers-Danlos syndrome (EDS). The small number of reported patients limits thorough investigation of this newly identified syndrome, currently coined as myopathic EDS. METHODS: DNA from 78 genetically unresolved patients fulfilling the clinical criteria for myopathic EDS was sequenced using a next-generation panel of COL12A1, COL6A1, COL6A2, and COL6A3. RESULTS: Among this cohort, we identified four pathogenic heterozygous in-frame exon skipping (∆) defects in COL12A1, clustering to the thrombospondin N-terminal region and the adjacent collagenous domain (Δ52, Δ53, Δ54, and Δ56 respectively), one heterozygous COL12A1 arginine-to-cysteine substitution of unclear significance (p.(Arg1863Cys)), and compound heterozygous pathogenic COL6A1 variants (c.[98-6G>A];[301C>T]) in one proband. Variant-specific intracellular accumulation of collagen XII chains, extracellular overmodification of the long isoform and near-absence of the short isoform of collagen XII, and extracellular decrease of decorin and tenascin-X were observed for the COL12A1 variants. In contrast, the COL6A1 variants abolished collagen VI and V deposition and increased tenascin-X levels. CONCLUSION: Our data further support the significant clinical overlap between myopathic EDS and collagen VI-related myopathies, and emphasize the variant-specific consequences of collagen XII defects.

The insoluble TGFBIp fraction of the cornea is covalently linked via a disulfide bond to type XII collagen.

TGFBIp, also known as keratoepithelin and ßig-h3, is among the most abundant proteins in the human cornea, and approximately 60% is associated with the insoluble fraction following extraction in sodium dodecyl sulfate (SDS) sample buffer. TGFBIp is of particular interest because a wide range of mutations causes amyloid or fuchsinophilic crystalloid deposits in the cornea leading to visual impairment. We show that the SDS-insoluble fraction of TGFBIp from porcine and human corneas is covalently linked via a reducible bond to the NC3 domain of type XII collagen in a TGFBIp:type XII collagen stoichiometric ratio of 2:1. Because type XII collagen is anchored to striated collagen fibers of the extracellular matrix, its interaction with TGFBIp is likely to provide anchoring for cells to the extracellular matrix through the integrin binding capability of TGFBIp. Furthermore, the TGFBIp-type XII collagen molecule will affect our understanding of the molecular pathogenesis of the TGFBI-linked corneal dystrophies.

Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

Zebrafish collagen XII is present in embryonic connective tissue sheaths (fascia) and basement membranes.

Connective tissues ensure the cohesion of the tissues of the body, but also form specialized structures such as tendon and bone. Collagen XII may enhance the stability of connective tissues by bridging collagen fibrils, but its function is still unclear. Here, we used the zebrafish model to visualize its expression pattern in the whole organism. The zebrafish col12a1 gene is homologous to the small isoform of the tetrapod col12a1 gene. In agreement with the biochemical data reported for the small isoform, the zebrafish collagen XII alpha1 chain was characterized as a collagenase sensitive band migrating at approximately 200 kDa. Using newly generated polyclonal antibodies and anti-sense probes, we performed a comprehensive analysis of its expression in developing zebrafish. Collagen XII exhibited a much broader expression pattern than previously thought: it was ubiquitously expressed in the connective tissue sheaths (fascia) that encase the tissues and organs of the body. For example, it was found in sclera, meninges, epimysia and horizontal and vertical myosepta. Collagen XII was also detected in head mesenchyme, pharyngeal arches and within the spinal cord, where it was first expressed within and then at the lateral borders of the floor plate and at the dorsal midline. Furthermore, double immunofluorescence staining with laminin and immunogold electron microscopy revealed that collagen XII is associated with basement membranes. These data suggest that collagen XII is implicated in tissue cohesion by stabilizing fascia and by linking fascia to basement membranes.

Collagen XII interacts with avian tenascin-X through its NC3 domain.

Large oligomeric proteins often contain several binding sites for different molecules and can therefore induce formation of larger protein complexes. Collagen XII, a multidomain protein with a small collagenous region, interacts with fibrillar collagens through its C-terminal region. However, no interactions to other extracellular proteins have been identified involving the non-collagenous N-terminal NC3 domain. To further elucidate the components of protein complexes present close to collagen fibrils, different extracellular matrix proteins were tested for interaction in a solid phase assay. Binding to the NC3 domain of collagen XII was found for the avian homologue of tenascin-X that in humans is linked to Ehlers-Danlos disease. The binding was further characterized by surface plasmon resonance spectroscopy and supported by immunohistochemical co-localization in chick and mouse tissue. On the ultrastructural level, detection of collagen XII and tenascin-X by immunogold labeling confirmed this finding.

Tensile stress-dependent collagen XII and fibronectin production by fibroblasts requires separate pathways.

The intracellular mechanisms controlling mechano-dependent production of the two extracellular matrix proteins collagen XII and fibronectin were analyzed. Fibroblasts were cultured on either tensed (attached) or released (floating) collagen type-I gels, respectively. Collagen XII and fibronectin production was three- to fivefold higher under tensed than under released conditions. The general inhibitor of tyrosine phosphorylation, genistein (50 microM), and the MAP kinase inhibitor PD98059 (20 microM) selectively reduced collagen XII accumulation by tensed cultures. Addition of PD98059, but not genistein, downregulated tensile stress-induced tyrosine phosphorylation levels of ERK1/2 and focal adhesion kinase. Staurosporine as well as pretreatment with phorbol ester, which constitute means to downregulate classical and novel PKC activity, specifically blocked collagen XII but not fibronectin accumulation in tensed fibroblasts. ERK1/2 phosphorylation levels were not affected by staurosporine treatment. Chronic exposure to the protein kinase C inhibitors bisindolylmaleimide and calphostin C blocked increased production of both fibronectin and collagen XII from cells under tension. The data manifest that the mechano-dependent production of collagen XII and fibronectin requires separate pathways. The FAK-ERK1/2 pathway, a genistein-sensitive tyrosine kinase, and a distinct classical/novel PKC appear selectively required for increased production of collagen XII in cells under tensile stress, whereas fibronectin induction is regulated by a different PKC-dependent pathway.

Developmental distribution of collagen type XII in cartilage: association with articular cartilage and the growth plate.

Collagen type XII is a member of the fibril-associated collagens and is characterized by a short triple-helical domain with three extended noncollagenous NC3 domains. Previous studies suggested that collagen XII is a component of cartilage but little is known about its spatial-temporal distribution. This study uses a polyclonal antibody to the purified NC3 domain to investigate its developmental distribution in rat forelimb. Collagen XII was present at the joint interzone on embryonic day 16 (E16d) and restricted to the presumptive articular cartilage by E18d. Labeling of the articular surface intensified as development progressed postnatally (day 1 [1d] to 28d) and extended approximately six cell diameters deep. In juvenile rats, collagen XII antibodies also labeled the longitudinal and transverse septa of stacked chondrocytes in the growth plate. However, collagen XII was not associated at any developmental stage with the cartilaginous secondary ossification center and was only weakly expressed in epiphyseal cartilage. Ultrastructural localization of the NC3 domain epitope showed labeling of the surface of collagen II fibrils both in tissue and in isolated fibrils. The results presented provide further evidence that articular cartilage differs substantially from the underlying epiphyseal cartilage and that different chondrocytic developmental fates are reflected in the composition of their extracellular matrix starting early in development. In addition, collagen XII was distributed in areas of cartilage with more organized fibril orientation and may have a role in promoting alignment or stabilizing such an organization, thereby creating a matrix capable of withstanding load-bearing forces.